A wide variety of immunoassays have been based on competitive inhibition where analyte in the sample competes with a known amount of labeled analyte for a fixed amount of anti-analyte antibody. Enzyme labels are often used in competitive inhibition assays, where binding of anti-analyte antibody with an enzyme-analyte conjugate allows for separation of complexed and uncomplexed conjugate.
An assay has been reported, based on the ability of fragments of .beta.-galactosidase to complement and form active enzyme. In particular, a .beta.-galactosidase enzyme donor (ED) combines with a .beta.-galactosidase enzyme acceptor (EA) to form an active .beta.-galactosidase enzyme. Conjugating a small analyte or an analyte analogue to the ED at certain sites does not affect the rate of .beta.-galactosidase catalyzed activity. However, when the ED-analyte conjugate is bound by anti-analyte antibody, the enzyme-catalyzed reaction rate during the initial phase of the reaction is reduced. This reduction in enzyme-catalyzed reaction rate has been used to quantitate the determination of a plurality of analytes where ED-analyte conjugate present in an assay medium and analyte present in the sample compete for anti-analyte antibody prior to the addition of EA. The .beta.-galactosidase-catalyzed reaction rate increases as the amount of analyte present in the sample increases.
Although the assays can be performed in a hospital or other clinical laboratory setting, there is a need for simplified assay protocols which can be performed by relatively unskilled personnel and analyzed without the use of sophisticated equipment.